undergo a thorough blood pressure assessment and, if systemic hypertension is discovered, appropriate treatment should be initiated. Reduction of systemic blood pressure can result in the improvement of diabetic macular edema.30,31

Poiseuille’s law describes flow within a tube, where the resistance to flow is inversely related to the radius of the lumen raised to the fourth power. Under hypoxic conditions such as diabetic retinopathy, retinal arterioles dilate, decreasing resistance, resulting in

an increase in hydrostatic pressure in the retinal capil- laries.32–34 Retinal blood vessels have been observed to

progressively dilate as diabetic macular edema forms.35 Less commonly, macular edema is seen in the presence of ocular hypotony. Since tissue hydrostatic pressure equals intraocular pressure, hypotonous eyes develop macular edema due to a widened hydrostatic pressure gradient. Edema may improve if intraocular pressure rises.36 Interstitial hydrostatic pressure also decreases, with accompanying macular edema or subretinal fluid, when the retina

is subjected to vitreous traction.37

Oncotic pressure within the capillaries decreases with hypoalbuminemia. This is most commonly seen in patients with the nephrotic syndrome, protein-defi- ciency malnutrition, or severe liver disease. Oncotic pressure changes commonly accompany the formation of diabetic macular edema. Breakdown of the blood–retinal barrier (BRB) allows albumin to leak into the interstitial spaces, thereby raising tissue oncotic pressure. This draws fluid out of capillaries, across the vascular endothelium, resulting in macular edema. There is a strong correlation between increased VEGF levels, breakdown of the BRB, and macular edema.38 Endothelial damage, such as that seen with diabetic retinopathy and vein occlusions, compromises the intercellular tight junctions as well as the integrity of the barrier function of the cell membranes. This increased vascular porosity leads to oncotic pressure shifts and interstitial macular edema.

1.3Biochemical Basis for Diabetic Retinopathy

Diabetes causes similar microvascular abnormalities in the retinal vasculature, renal glomeruli, and vasa vasorum. In the early stages of diabetes,

chronic hyperglycemia results in blood flow alterations and increased vascular permeability. This is characterized by decreased activity of vasodilators such as nitric oxide and coexisting increased activity of vasoconstrictors such as angiotensin II and endothelin-1 with the release of vasopermeability augmenting cytokines such as VEGF. Resultant extracellular matrix abnormalities, both qualitative and quantitative, contribute to irreversible increases in vascular permeability. Microvascular cell loss occurs due to programmed cell death, the overproduction of extracellular matrix proteins and the deposition of periodic acid-Schiff-positive proteins induced by growth factors such as TGF-b, all of which subsequently lead to progressive capillary occlusion. Hyperglycemia decreases the production of endothelial and neuronal cell trophic factors leading to edema, ischemia, and hypoxia-driven neovascularization.39 Atherosclerosis in nondiabetic patients begins with endothelial dysfunction40 whereas in diabetics this seems to involve insulin resistance due to hyperglycemia.41

Four hypotheses have previously been advanced to explain the mechanism of hyperglycemia-induced microvascular damage. These are

1.Increased polyol pathway flux

2.Advanced glycation end products (AGEs)

3.Activation of protein kinase C (PKC)

4.Increased hexosamine pathway flux.

Specific inhibitors of aldose reductase, AGE formation, PKC activation, and the hexosamine pathway each prevent various diabetes-induced abnormalities, but no apparent common element was noted until the recent discovery that each causes overproduction of superoxide by the mitochondrial electron-transport chain39 (Fig. 1.6). It has been noted that both diabetes and hyperglycemia increase oxidative stress.42

To understand how hyperglycemia leads to an increase in reactive oxygen species (ROS) one must look at changes in the electron-transport chain within the mitochondria (see Fig. 1.6). Hyperglycemia, by causing overproduction of electron donors (NADH and FADH2) by the tricarboxylic acid (TCA) cycle,43 increases the proton gradient across the inner mitochondrial membrane. This prolongs the lifespan of electron-transport intermediates, such as ubisemiquinone, above a threshold

value, thereby significantly generating superoxide (Fig. 1.6). Two regulatory enzymes can be exploited to uncouple hyperglycemia-induced production of ROS. Upregulation of manganese superoxide dismutase (MnSOD) eliminates reactive oxygen species production; excess uncoupling protein-1 (UCP-1) eliminated the protein electrochemical gradient.44 Furthermore, overexpression of either MnSOD or UCP-1 prevented PKC activation, activation of the hexosamine pathway, AGE formation, and an increase in polyol pathway flux. This evidence strongly supports the belief that excessive superoxide is central to the unified theory of diabetic retinopathy.

Other experimental evidence links hyperglycemia, ROS, and the four above-mentioned biochemical pathways (see Fig. 1.7). Hyperglycemiainduced increase in ROS decreases glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity and, therefore, causes an increase in upstream glycolytic metabolites. This leads to an increase in the polyol pathway flux. Methylglyoxalderived AGE, the most common AGE resulting from hyperglycemia, probably results from increased triose phosphate levels. Triose phosphate levels rise with GAPDH inhibition by ROS.45 ROS-induced decreases in GAPDH activity causes a buildup of fructose-6-phosphate, the primary substrate for

the hexosamine pathway. Inhibition of GAPDH leads to elevated dihydroxyacetone phosphate levels, leading to increased DAG concentrations and activation of PKC.

Several experimental models have shown that the elevated MnSOD or UCP-1 activity prevents hyperglycemia-induced complications. Overexpression of either protein prevents monocyte adhesion to aortic endothelial cells,39 the hyperglycemiainduced decrease in eNOS activity,43 and collageninduced platelet aggregation and activation.46 Increased MnSOD activity prevents an increase in collagen synthesis47 and decreases programmed cell death induced by hyperglycemia.

Since considerable clinical research effort continues to focus on decreasing diabetic complications by minimizing changes in the four-affected pathways, further discussion of the pertinent biochemistry is warranted.

1.3.1 Increased Polyol Pathway Flux

Aldose reductase, the first enzyme in the polyol pathway, has a low affinity for glucose at normal concentrations. In hyperglycemia, however, the

elevated glucose levels result in increased conversion into sorbitol with associated decreases in NADPH. Sorbitol is then oxidized to fructose with NADH reconstitution (Fig. 1.7). It has been proposed that sorbitol oxygenation increases the NADH/NAD+ ratio in the cytosol, thereby inhibiting activity of glyceraldehyde-3-aldehyde dehydrogenase (GAPDH). This leads to increasing concentrations of triose phosphate,48 which increases the formation of methylglyoxal – a precursor of AGEs – and diacylglycerol, thus activating PKC. Reduction of glucose to sorbitol consumes NADPH; since NADPH is required for regeneration of reduced glutathione, this could exacerbate oxidative stress. Attempts to inhibit the polyol pathway in vivo have yielded mixed results. A 5-year study in diabetic dogs prevented diabetic neuropathy but failed to prevent retinopathy.49 Zenarestat, an aldose reductase inhibitor, demonstrated a positive effect on diabetic neuropathy in humans.50

1.3.2Advanced Glycation End Products (AGEs)

Intracellular hyperglycemia is the inciting event for the formation of AGEs, which are found in increased concentrations in diabetic retinal blood

vessels51 and glomeruli.52 They arise from the intracellular auto-oxidation of glucose to glyoxal, the decomposition of the Amadori product (glucose-derived 1 amino-1-deoxyfructose lysine adducts) to 3-deoxyglu- cosone, and the fragmentation of glyceraldehyde-3- phosphate and dihydroxyacetone phosphate to methylglyoxal, all of which react with amino groups

of intracellular and extracellular proteins to form AGEs (see Fig. 1.8).53–55 The AGE inhibitor amino-

guanidine partially prevented microvascular damage in animal models56 and lowered urinary protein and slowed progression of retinopathy in humans.57

The production of intracellular AGE precursors damages target cells by modifying proteins and altering their function. This changes extracellular matrix components and integrins and modifies plasma proteins that bind to AGE receptors. The end result is receptor-mediated production of reactive oxygen species.

AGE formation alters the properties of several extracellular matrix proteins. Crosslinking by AGEs induces an expansion of the molecular packing of type I collagen, thereby altering the function of vessels.58 AGEs alter type IV collagen from basement membranes.59 AGE formation on laminin causes decreased polymer self-assembly, decreased binding to type IV collagen, and decreased binding to heparin sulfate proteoglycan.60

AGE formation on extracellular matrix interferes with matrix–cell interactions. Modification of type IV collagen-binding domains decreases endothelial cell adhesion. Modification of a 6-amino acid growth-promoting sequence in the A chain of laminin reduces neurite outgrowth.61

Several cell-associated-binding proteins for AGEs have been identified: OST-48, 80K-H, galec- tin-3, macrophage scavenger receptor type II, and RAGE. They mediate the long-term effects of AGEs on macrophages, glomerular mesangial cells, and vascular endothelial cells. Their effects include the expression of cytokines and growth factors (interleukin-1, insulin-like growth factor I, tumor necrosis factor-a, TGF-b, macrophage col- ony-stimulating factor, granulocyte–macrophage colony-stimulating factor, and platelet-derived growth factor) by macrophages and mesangial cells, and the expression of pro-coagulatory and pro-inflammatory molecules (thrombomodulin, tissue factor, and VCAM-1) by endothelial cells. The binding of ligands to endothelial AGE receptors mediates the capillary wall hyperpermeability

induced by VEGF.62 Blockage of RAGE suppressed macrovascular disease in an atherosclero- sis-prone type 1 diabetic mouse model. RAGE blockade also inhibited the development of diabetic nephropathy and periodontal disease.

1.3.3 Activation of Protein Kinase C (PKC)

Protein kinase C is a family of at least 11 isoforms, 9 of which are activated by the lipid second messenger diacylglycerol (DAG). Intracellular hyperglycemia increases DAG in both the retina and renal glomeruli by increasing synthesis from dihydroxyacetone phosphate (see Fig. 1.9).63 This, in turn, activates PKC in vascular cells, retina, and glomeruli. Hyperglycemia also activates PKC isoforms indirectly through ligation of AGE receptors64 and via increased activity of the polyol pathway.65 Activation of PKC-b isoforms mediates retinal and renal blood flow abnormalities by depressing nitric oxide production and increasing endothelin-1